Currently, worldwide annual production of methionine is about 500,000 tons. Methionine is the first limiting amino acid in livestock of poultry feed and due to this, mainly applied as feed supplement. In contrast to other industrial amino acids, methionine is almost exclusively applied as a racemate produced by chemical synthesis. Since animals can metabolise both stereoisomers of methionine, direct feed of the chemically produced racemic mixture is possible (D'Mello and Lewis, Effect of Nutrition Deficiencies in Animals: Amino Acids, Rechgigl (Ed.), CRC Handbook Series in Nutrition and Food, 441-490, 1978).
However, there is still a great interest in replacing the existing chemical production by a biotechnological process. This is due to the fact that at lower levels of supplementation L-methionine is a better source of sulfur amino acids than D-methionine (Katz et al., (1975) Poult. Sci., 545: 1667-74). Moreover, the chemical process uses rather hazardous chemicals and produces substantial waste streams. All these disadvantages of chemical production could be avoided by an efficient biotechnological process.
For other amino acids such as glutamate, fermentation production methods are known. For these purposes, certain microorganisms such as Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum) have proven to be particularly suited. The production of amino acids by fermentation also has the particular advantage that only L-amino acids are produced. Further, environmentally problematic chemicals such as solvents, etc. which are used in chemical synthesis are avoided. However, fermentative production of methionine by microorganisms will only be an alternative to chemical synthesis if it allows for the production of methionine on a commercial scale at a price comparable to that of chemical production.
Hence, the production of L-methionine through large-scale culture of bacteria developed to produce and secrete large quantities of this molecule is a desirable goal. Improvements to the process can relate to fermentation parameters, such as stirring and supply of oxygen, or the composition of the nutrient media, such as the sugar concentration during fermentation, or the working up of the product by, for instance, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis and mutant selection are also used to improve the output properties of these methionine-producing microorganisms. High production strains which are resistant to antimetabolites or which are auxotrophic for metabolites of regulatory importance are obtained in this manner.
Recombinant DNA technology has also been employed for some years for improving microorganism strains which produce L-amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
Rückert et al. (Journal of Biotechnology (2003), 104: 213-228) provide an analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum. Known functions of MetZ (also known as MetY) and MetB could be confirmed and MetC (also known as AecD) was proven to be a cystathionine-β-lyase. Further, MetE and MetH, which catalyse the conversion of L-homocysteine to L-methionine, were identified in this study.
WO 02/097096 discloses nucleotide sequences from coryneform bacteria which code for the McbR repressor gene (also known as MetD) and processes for the preparation of amino acids using bacteria in which this McbR repressor gene is attenuated. According to WO 02/097096, the attenuation of the transcriptional regulator McbR improves the production of L-methionine in coryneform bacteria. It is further described in WO 02/097096 that, in addition to the attenuation of the McbR repressor gene, enhancing or overexpressing the MetB gene which codes for cystathionine-γ-synthase is preferred for the preparation of L-methionine.
Selection of strains improved for the production of a particular molecule is a time-consuming and difficult process. Therefore, there is still a great need for microorganisms which efficiently produce L-methionine and/or have significantly increased contents of L-methionine which can be utilized for obtaining methionine.